During embryonic development of the nematode Caenorhabditis elegans and many other organisms, a stereotyped pattern of cell cleavages gives rise to a set of founder cells with differing developmental potential. Cell divisions are either proliferative (with daughters having the same developmental potential and size) or determinative (with daughters having differing developmental potential and often differing sizes). Proliferative divisions exhibit a standard pattern of successively orthogonal cleavage planes, whereas determinative divisions are usually in the same plane as the previous division. Mechanisms have been demonstrated that align the mitotic apparatus in C. elegans along a pre- formed axis during the first few determinative divisions of the embryo. In addition, after alignment, the mitotic apparatus often becomes displaced along its length to become eccentrically positioned in the cell, giving rise to an asymmetric division (the cleavage plane bisects the mitotic apparatus). It is proposed to study the mechanisms that specify the angular alignment and axial position of the mitotic apparatus in cells undergoing determinative divisions, thereby gaining an understanding of the mechanisms that define cleavage planes. These mechanisms are likely to be central to the process of cellular differentiation and are therefore likely to be relevant to pathologies such as cancer in which this process has become perturbed. Fluorescent analogues of tubulin and actin will be injected into syncytial oocytes so that they become incorporated into fertilized eggs. Low-dose in vivo confocal fluorescence microscopy will be used to characterize the dynamics of these cytoskeletal components as the mitotic apparatus becomes aligned during the first few divisions of the embryo. In particular, the interactions of astral microtubules of the mitotic apparatus with the cortex will be investigated. A more detailed picture of the cytoskeletal organization at defined stages will be obtained by means of correlative electron microscopy. These techniques will be applied to both wild-type embryos and to embryos of known mutants with altered early cleavage patterns. In addition, mutants will be isolated that specifically disrupt aspects of the spindle alignment process.